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Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 1075-1079

Is the Wnt/β catenin signalling pathway activated in Seminoma?: An immunohistochemical study

1 Cytology, Department of Clinical Biochemistry, Clinical Hospital (UBA), INFIBIOC-Argentina, Argentina
2 Department of Pathology, Freiburg University Medical Center, Freiburg, Germany
3 Laboratory of Pathology, Ramos Mejía Hospital, Argentina
4 Department of Pathology, Clinical Hospital (UBA), C.A.B.A.-Argentina, Argentina

Date of Web Publication25-Jul-2016

Correspondence Address:
Palaoro Luis Alberto
Avda Forest 1318 4° B - (1427), Continental Automated Buildings Association, Buenos Aires
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Source of Support: This work was supported by UBACYT, Bs Aires University, Argentina, Conflict of Interest: None

DOI: 10.4103/0973-1482.147392

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 > Abstract 

Background: The loss of reduction of some adhesion molecules is associated with the invasive phenotype of carcinomas. The aim of this paper was to study the expression of some proteins related to the Wnt/β catenin signalling pathway in seminomas by immunohistochemical techniques in order to assess the contribution of this pathway to the tumoral development.
Materials and Methods: Immunohistochemistry for E-cadherin, β-catenin, vimentin, C-Myc, cyclin D1 (CD1) and placental alkaline phosphatase. (PALP) was carried out in 24 archival tissue blocks of seminomas. Two cellular lines were used as E-cadherin and β-catenin controls. (JKT-1 and TCam-2).
Results: E-cadherin was positive in two seminomas and in one carcinoma in situ. (CIS), showing a membranous pattern. βcatenin was principally expressed in Sertoli cells, in some malignant gonocytes of CIS and in the membranes of seminomatous cells. No β-catenin immunostaining was detectable in the cytoplasm and the nuclei of neoplastic cells. Seminomas were weakly possitive for vimentin in 11/24 cases. None of the tumors displayed expression of C-Myc or CD1.
Conclusions: The results does not indicate the activation of the Wnt pathway, due to the lack of vimentin expression in 13/24 seminomas, the low expression in the rest of the cases, the lack of β-catenin in nuclei and the absence of CD1 and C-Myc expression. Further work is needed to confirm these observations and to test other signaling pathways in seminomas.

Keywords: β catenin, catenin signalling pathway, seminoma, vimentin, Wnt/β

How to cite this article:
Fernando G, Paul F, Laura J, Alejandra AM, Gabriela M, Alberto PL. Is the Wnt/β catenin signalling pathway activated in Seminoma?: An immunohistochemical study. J Can Res Ther 2016;12:1075-9

How to cite this URL:
Fernando G, Paul F, Laura J, Alejandra AM, Gabriela M, Alberto PL. Is the Wnt/β catenin signalling pathway activated in Seminoma?: An immunohistochemical study. J Can Res Ther [serial online] 2016 [cited 2020 Aug 10];12:1075-9. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/1075/147392

 > Introduction Top

Germ cell tumors are believed to arise from a common cell, the primordial germ cell.[1] It is considered that both seminomatous and nonseminomatous germ cell tumors develop through a stage of carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU). The switch from pre-invasive CIS to invasive tumor takes place in situ by tubular enlargement followed by sertoli cell degeneration.[2]

The pathogenesis of these different germ tumors remain poorly defined.[3],[4],[5],[6]

Loss or reduction of certain adhesion molecules, such as E-cadherin, appears to be associated with the development of human carcinomas and the change to an invasive and metastatic phenotype.[7]

Abnormal E-cadherin expression was detected in many carcinomas (including breast, stomach, pancreas),[8],[9],[10] but in the case of germ cell tumor the results are yet scanty and conflicting.[11],[12],[13],[14]

The aim of this study was to investigate the expression of E-cadherin, β-catenin, and same proteins related to the Wnt/β catenin signalling pathway in seminomas by immunohistochemistry, in order to estimate the contribution of this pathway to the development of seminomas.

 > Materials and Methods Top

Twenty-four formalin-fixed paraffin-embedded archival tissue blocks of seminomas from the Department of Pathology of two hospitals were used in this study. Of them, 14 were associated to CIS. A 3-5 μm thick tissue sections each (about 15 of each smear) were deparaffinized in xylene in two steps: 30 minutes at 56°C and 20 minutes at room temperature. Then were rehydrated through graded concentrations of ethylic alcohol and placed in citrate buffer pH 6.0 for antigen retrieval for 30 minutes at 96-98°C. Then, the slides were cooled in the same buffer for 20 minutes and placed in phosphate buffer saline (PBS) pH 7.2. The samples were incubated with several primary antibodies: 30 minutes for placental alkaline phosphatase (PALP; Dako, Carpinteria, CA, USA, dilution 1:250), β-catenin (BD Biosciences, Denver Co, USA; dilution 1:100), vimentin (Dako, Carpinteria, CA, USA; dilution 1:400) C-Myc (Novus Biologicals, Littleton, USA; dilution 1:50), E-cadherin (Dako, Carpinteria, CA, USA; dilution 1:200) and 60 minutes for cyclin D1 (CD1; Dako, Carpinteria, CA, USA, dilution 1: 50). After washing with PBS, the slides were incubated with biotinylated-secondary antibody for 20 minutes, rinsed with PBS and covered with streptavidin-alcaline phosphatase. It was rinsed again and finally incubated with the developing buffer of Fast Red TR for 10 minutes. A qualitative evaluation of the staining intensity was recorded as negative, low positive and positive. Low positive was considered if less than 20% of cells were immunostained, and positive if more than 20% of cells were stained. Membranous or cytoplasmic expression for all the markers was recorded. Two cellular lines were used as E-cadherin and β-catenin controls (JKT-1 and Tcam 2: Negative and positive controls respectively).

 > Results Top

E-cadherin was not expressed in sertoli and interstitial cells, but was positive in two seminomas and one CIS (about 30% of the seminomatous cells expressed this marker), and low positive in three seminomas (about 10% of the seminomatous cells expressed this marker), showing a membranous pattern in all cases. The rest of the cells, such as lymphocytes or fibroblasts were negative, as expected [Figure 1].
Figure 1: Low and membranous expression of E-cadherin in seminoma (Immunostaining ×400)

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β-catenin was principally expressed in sertoli cells and in some malignant gonocytes of CIS (membranous pattern, whereas few gonocytes showed aberrant cytoplasmic stained) [Figure 2] and it was detected in the membranes of seminomatous cells in all the smears (24/24) [Figure 3]. In two biopsies the expression of this marker was strongly positive in the cytoplasm of sertoli cells. No β-catenin immunostaining was detectable in the cytoplasms and nuclei of neoplastic cells. Endothelial cells of some blood vessels showed weak reactivity for β-Catenin. Some stromal cells (four cases) and leydig cells (one case) showed β-catenin expression [Figure 4].
Figure 2: CIS showing β-catenin expression in sertoli cells and in some atypical gonocytes (Immunostaining ×400)

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Figure 3: Membranous pattern of β-catenin in seminoma (Immunostaining ×400)

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Figure 4: Leydig cells (center) expressing β-catenin (Immunostaining ×400)

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Eigth out of 14 CIS showed immunostaining of vimentin only in sertoli cells [Figure 5] and [Figure 6], while seminomas presented low positive staining for vimentin in 11/24 cases [Figure 7]. All controls showed positive vimentin staining in stromal cells: Lymphocytes, endothelial cells and fibroblasts, and it was used as internal positive control [Figure 8]. Four CIS and 15 seminomas strongly expressed cytoplasmic PALP, while, as expected, the rest of the tissue, such as the lymphocytes surrounding the tumor cells were negative for this marker [Figure 9].
Figure 5: CIS showing cytoplasmic vimentin in sertoli cells. Atypical gonocytes are negative (Immunostaining ×400)

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Figure 6: Strong expression of vimentin in sertoli cells. Note the lack of staining in atypical gonocytes (Immunostaining ×400)

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Figure 7: Low expression of vimentin in seminoma (Immunostaining ×400)

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Figure 8: Vimentin positive in stromal cells, and negative in seminomatous cells (Immunostaining ×400)

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Figure 9: Seminoma expressing placental alkaline phosphatase (Immunostaining ×400)

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There were no tumor cells with C-Myc or CD1 expression [Table 1].
Table 1: Immunohistochemistry of invasive seminomas and CIS

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The cell line JKT-1 was negative for E-cadherin and β-catenin, while TCam-2 cells showed a positive membranous pattern for both markers.

 > Discussion Top

E-cadherin and other proteins such as β-catenin form part of the complex necessary for the cellular adhesion,[15] while the loss or down regulation of E-cadherin expression results in the development of cell motility and invasiveness.[16]

Several oncogenes and tumour suppressor genes have been implicated in germ cell tumor origin, but until now there is not a clear explanation for the self-renewal or differentiation into multiple cell types similar to stem cells.[3],[4] Several pathways control these processes including wnt/β catenin signalling, Notch signalling, the hedgehog signalling pathway or the NF-κB Pathway.[5],[6]

The Wnt/β-catenin signalling pathway is important in the regulation of differentiation and proliferation of stem cells,[17] and their activation is related to the development of several cancers.[18]

In the canonical signalling pathway, the accumulation of β-catenin within the nucleous is a necessary step for the transcription of specific genes related to the tumorigenesis. Changes in E-cadherin expression can induce the process called epithelial-mesenchymal transition (EMT),[18],[19],[20] which shows, among other changes, the translocation of β-catenin to the nucleus to engage the activation of the Wnt signalling pathway.[21]

Under normal conditions, when the cell to cell contact prevails there is no Wnt ligand signalling, β-catenin is phosphorylated in the cytoplasm and therefore is ubiquitinizated and degradated by the proteosome. When Wnt is activated, β-catenin accumulates within the nucleus and interacts with the T-cell factor–lymphoid enhancer binding factor (Tcf-Lef) in order to regulate the transcription of the particular genes, such as C-Myc, CD1, vimentin, matrix metalloproteinase-7 (MMP7), urokinase-type plasminogen activator receptor (UPAR), CD44, Jun A-P1, Fos-Related Antigen-1 (FRA-1), Peroxisome Proliferative Activated Receptor-Delta (PPARD), Transcription Factor-1 (TCF-1) (), Fibronectin, Slug, Gastrin, Cyclooxygenase-2 (COX2) and the Gamma-2 chain of Laminin 5.[22],[23]

In the present study, expression of E-cadherin was low or absent in the majority of the CIS and seminomas and was accompanied by a high β-catenin staining, although its localization appeared to be restricted in cellular membranes. The negative staining for E-cadherin was also reported by others, who showed their absence in intratubular and invasive seminomas.[12]

β-catenin protein expression has been demonstrated in frozen tissue of testicular tumors,[11] but in that study the intensity or localization of the staining (nuclear, membranous or cytoplasmic) was not demonstrated. On the other hand, it is interesting that in ovarian dysgerminomas (the female counterpart of testicular seminomas) significant β-catenin staining by immunohistochemistry in deparaffinized sections has not been demonstrated.[20]

Therefore, our results are not in accordance with that report,[20] because we observed β-catenin staining in seminomatous cells, but with a membranous pattern and never within the nuclei (24/24 cases). In a previous paper, β-catenin was negative in invasive seminomas,[12] but in this study a portion of the samples came from patients treated with chemotherapy, so it is difficult to make a comparison between these results and ours. The weak reactivity for β-catenin in the endothelium of blood vessels could be related to the modification of the levels of this protein during the loss and restoration of adherent junctions. Whether this process is related to the evolution of seminomas and its spread, is at present a matter of speculation.[24]

In addition, there is no clear explanation for the expression of β-catenin that we have observed in leydig cells in one of the samples. Recently, it has being reported the elevated expression of this marker in rat leydig cells with stimulated testosterone synthesis.[25]

Expression of PALP in the majority of the seminomas confirmed as expected the diagnosis of the tumors.[26]

An aberrant expression of E-cadherin has been reported in 19%[13] and 18%[11] of seminomas. In order to relate E-cadherin expression to the Wnt signalling pathway, we performed immunostaining for vimentin. Vimentin is one of the markers of Epithelial-mesenchymal Transition (EMT), as a consequence of the transcription of several genes activated by β-catenin and the TCF-LEF transcription factors. However our results seem not indicate the activation of the Wnt pathway due to the lack of vimentin expression in 13/24 seminomas (with low expression in the rest of the cases) and the lack of β-catenin nuclear staining. These results are in agreement with the absence of CD1 and C-Myc expression in all sections, two genes regulated by the Wnt/β-catenin pathway.

In summary we conclude that the Wnt/β-Catenin signalling pathway does not contribute to the pathogenesis of classical seminomas. We propose that vimentin expression (although not very intense) in some sections could be explained by the activation of some other signalling pathways, such as the Ras-MAPK pathway, which is deregulated in more than 30% of human cancers.[27]

The activation of GTPase HRas has been shown to induce EMT like changes in non-malignant MCF10A breast epithelial cells in recent studies.[28]

These changes include downregulation of E-cadherin and β-catenin as well as upregulation of the receptor tyrosine kinase AXL, the transcription factor SLUG and vimentin.[29]

Thus, the Ras-MAPK pathway could be one of the pathways that should be further investigated for the analysis of the pathogenesis of seminomatous germ tumors.

 > Ethics Statement Top

All tissue samples were collected for histologic examination and diagnostic purposes and were thoroughly anonymized for the use in this study. Thus no informed consent was needed. (National Law on Protection of Personal Data (No. 25326). This study was approved by the Institutional Review Board of our Hospital. Researchers respect the Helsinky Declaration in its latest version (World Medical Association Declaration of Helsinki 2013).

 > References Top

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Donner J, Kliesch S, Brehm R, Bergmann M. From carcinoma in situ to testicular germ cell tumour. APMIS 2004;112:79-88.  Back to cited text no. 2
Chaganti RS, Houldsworth J. Genetics and biology of adult human male germ cell tumors. Cancer Res 2000;60:1475-82.  Back to cited text no. 3
Schneider DT, Schuster AE, Fritsch MK, Calaminus G, Harms D, Göbel U, et al. Genetic analysis of childhood germ cell tumors with comparative genomic hybridization. Klin Padiatr 2001;213:204-11.  Back to cited text no. 4
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Batistatou A, Scopa CD, Ravazoula P, Nakanishi Y, Peschos D, Agnantis NJ, et al. Involvement of dysadherin and E-cadherin in the development of testicular tumours. Br J Cancer 2005;93:1382-7.  Back to cited text no. 13
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1]


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